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2012 2nd International Conference on Biotechnology and Environment Management
IPCBEE vol. 42 (2012) © (2012) IACSIT Press, Singapore
DOI: 10.7763/IPCBEE. 2012. V42. 7
Green Technology for One-step Production of Alkyl Glycosides Using
Cyclodextrin Glycosyltransferase from Paenibacillus sp.RB01
Kasinee Katelakha Piamsook Pongsawasdi and Manchumas Hengsakul Prousoontorn 
Starch and Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn
University, Bangkok 10330, Thailand.
Abstract. Biological production of alkyl glycosides were performed using enzyme catalytic method instead
of the use of chemical catalyst. Cyclodextrin glycosyltransferase (CGTase) from Paenibacillus sp.RB01 was
used to catalyze the transglycosylation reaction from a glycosyl donor to an appropriate alcohol acceptor
including primary, secondary and tertiary alcohols. The reaction mixtures containing 0.6% (w/v) cyclodextrin (-CD), 2-30 % (v/v) of various alcohols and 200 U/mL of CGTase in 50 mM acetate buffer pH
6.0 were performed at 40 C for 24 hours with continuous shaking. The effect of alcohols on the
transglycosylation activity of CGTase was assayed. It was found that the alcohol length and concentration
had significant effect on the enzyme activity. The highest medium chain alkyl glycoside yield was obtained
when isobutanol (5% v/v) was used as a glycosyl acceptor. Products were checked to confirm that they were
glycoside derivatives by the treatment with glucoamylase and -glucosidase. The result obtained showed that
the product was a glycoside derivative which had an -linkage between isobutanol and glucose residue.
Various carbohydrate sources could be used as glycosyl donor for the process of the alkyl glycoside
production.
Keywords: Alkyl glycoside, Cyclodextrin glycosyltransferase, Transglycosylation
1. Introduction
Green Technology has been mentioned in the past few years due to the environmental problems are still
increasing as a result of the environmental care products demand increased. The biological processes have
thus been developed for any manufacturing steps.
Alkyl glycosides are classified as a non-ionic surfactant which is produced from the reactions of fatty
alcohols and saccharides. Therefore, they are rapidly biodegradable and non-toxic. Their structures are
amphipathic molecules consisting of one or several units of sugar head group connected via an ether bond
with hydrophobic hydrocarbon chain. They have good emulsification activity and wetting properties.
Furthermore, the sugar head group could ionize with no charge. Thus, they have potential use as effective
agents in membrane solubilization and also in food and cosmetic industries [1]. Enzymatic synthetic method
is advantageous over the chemical ones, as they ensure the stereo-selective obtaining of the product.
Although it has been shown that their chemical synthesis was well-developed, the method still employs
toxic and expensive compounds to yield
α and β-configuration products via several steps. Hence, the biological process of one-step production of
alkyl glycosides has been developed using enzyme catalyzed glycosylation reaction [2, 3]. On the other hand,
reverse hydrolysis reaction was also employed by controlling water activity in the reactions [4].
Long chain alkyl glycosides have higher surfactant activity than those of the short chain ones. Therefore,
they will undoubtedly constitute an important part not only in industrial chemicals widely used in almost
every sector of various industries but also in household uses [5]. In 2011, short-chain primary α-alkyl

Corresponding author. Tel.: +6622185437; fax: +6622185418
E-mail address: [email protected]
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glycosides were successfully synthesized through transglycosylation reaction by cyclodextrin
glycosyltransferase (CGTase, E.C. 2.4.1.19) from Paenibacillus sp. RB01 which was isolated from hot
spring area in Thailand [6]. The use of CGTase for alkyl glycoside synthesis is of great interest due to the
fact that a glycoside product containing more than one sugar residues could be synthesized. On the other
hand, the reaction catalyzed by glycohydrolases was able to transfer only one monosaccharide unit.
In this work, we demonstrated the synthesis of alkyl glycosides from miscible and immiscible alcohols
including primary, secondary and tertiary ones through one-step system using CGTase from Paenibacillus sp.
RB01. Effect of alcohols on the transglycosylation ability of the enzyme was investigated and the product
obtained was checked for the anomeric structure by treatment with hydrolytic enzymes. The ability to use
various donor substrates was also determined.
2. Materials and Methods
2.1. Enzymes and Chemicals
β-Cyclodextrin, methyl-α-D-glucopyranoside and α-glucosidase were obtained from Sigma-Aldrich
Chemical Co., USA. Glucoamylase (amyloglucosidase) was from Aspergillus niger and 2-hexanol were
purchased from Fluka, Switzerland. Soluble starch (synthesis grade from potato) and flomax® were from
Scharlau Chemie S.A., Spain and national starch USA, respectively. Silica gel 60 F254 aluminium sheets (0.2
mm), methanol, ethanol, cyclohexanol and 1-octanol were obtained from Merck, Germany. 1-propanol, 2propanol, and 1-butanol were from Carlo Erba Reagents, Italy. Isobutanol, 2-butanol, 1-pentanol and 1heptanol were purchased from BDH Chemical, England. 2-pentanol was obtained from Lab Scan, Thailand.
Other chemicals were of analytical grade.
2.2. Synthesis of Various Alkyl Glycosides by Cyclodextrin Glycosyltransferase
CGTase from Paenibacillus sp. RB01 was purified by starch adsorption technique prior to be used as a
biocatalyst in the transglycosylation reaction for the synthesis of alkyl glycosides [7]. The reaction mixture
containing 0.6% (w/v) β-CD and different alcohols as acceptors was incubated with CGTase (200 U/mL) in
50 mM acetate buffer pH 6.0 at 40°C for 24 hours with continuous shaking. The effect of alkyl chain length
and concentration on CGTase were carried out using phenolphthalein method [8]. The relative coupling
activity was calculated from the disappearance of β-CD substrate. The decrease in absorbance at 550 nm was
measured and conversion of ∆A550 to µmoles of β-CD was quantitated using β-CD phenolphthalein
calibration curve. The β-CD disappearance in buffer was set as 100 percent.
2.3. TLC Analysis
The reaction mixture was evaporated at 30°C for 3 hours. The evaporated sample was adjusted to 100 μL
with distilled water. The product were analyzed by applying 5 μL of each sample on siliga gel 60 F254
aluminium sheets with ethyl acetate: acetic acid: water (3:1:1) as a mobile phase. The compounds were
visualized by spraying with sulfuric: methanol (1:2 by vol) and heating at 110°C for 10 minutes. The amount
of product was determined from spot intensities using GeneTools program of SYNGENE Synoptic Ltd.,
England.
2.4. Preliminary Characterization of Glycoside Product Structure
Alkyl glycoside from the CGTase-catalyzed reaction was further hydrolyzed by hydrolytic enzyme to
confirm that the product was glycoside derivative. The reaction mixture was evaporated to remove the
alcohol substrate before treatment with glucoamylase (final concentration of 40 U/mL) for 1 hour. The
reaction was stopped by boiling at 100°C for 10 minutes. The alpha linkage between alcohol and glucose
residue was also checked by treatment the transglycosylation reaction with α-glucosidase (final concentration
of 40 U/mL) at 40°C for 3 hours. The result obtained was checked with TLC.
2.5. Donor Specificity
To find an appropriate donor, various carbohydrates including β-CD, raw starch, and soluble starch both
from potato and cassava - flomax® were used as a glucosyl donor. Various donors were used at the final
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concentration of 0.6% (w/v) under the optimal condition for CGTase as described previously. Alkyl
glycoside products produced from each donor were quantitated from spot intensities on the TLC plate.
3. Results and Discussion
3.1. The Effect of Alcohol Length and Concentration on the Production of Alkyl Glycosides
The effect of alcohols on coupling activity of CGTase was investigated in buffer and in a mixture of
buffer and alcohol solution with varying alcohol length and concentration at the optimum pH (6.0) of the
enzyme for 24 hours.
Fig. 1: Relative coupling activity of CGTase in alcohol solvent mixtures. Residual activity (%) was relative to the
coupling activity of CGTase incubated with 0.6% (w/v) β-CD and various concentration of alcohol in 50 mM acetate
buffer solution (pH 6.0) at 40ºC for 24 hours. All values were average from two separate experiments.
As can be seen in Fig. 1, when the concentration of alcohol increased, the activity of the enzyme was
dramatically decreased. Medium and long chain alcohols at the concentration above 5% (v/v) are non soluble
so shaking is needed and might cause the reaction to proceed non-uniformly. Thus, the activity of CGTase in
biphasic system still remained whereas the coupling activity of the enzyme in monophasic system
dramatically decreased. These results showed that the alcohol length and concentration had significant effect
on CGTase activity. Cyclohexanol has been shown to compete with phenolphthalein to form complexation
with hydrophobic cavity of CD. Hence, the complexation between phenolphthalein and CD appeared to be
strongly effected [9]. Although branched chain primary alcohols had more effect on enzyme activity but they
were more soluble than that of normal chain primary alcohol so the production yield was found to be higher.
From these results, isobutanol was chosen as an appropriate acceptor. The TLC chromatogram of the
CGTase catalyzed the transfer of glucose from β-CD to various alcohols are shown in Fig. 2. By adsorption
chromatographic mechanism, the expected alkyl glycoside products with less polarity due to the length of
alkyl chains migrated with larger Rf values than those of sugar in the non-polar mobile phase. The number of
spots detected was dependent on polarity of the products which correlated with the length as well as the
conformation of alkyl chain and the number of glucose residues. The product obtained from the CGTasecatalyzed transglycosylation reaction was more diversified than those from action of β-glucosidase [10].
3.2. Preliminary Characterization of Reaction Products Using Amylolytic Enzymes
Reaction mixture of selected acceptor was further hydrolyzed with glucoamylase–an enzyme which
hydrolyzes glycosidic linkage between glucose residues and α-glucosidase to confirm the linkage between
alcohol acceptor and glucose. Fig. 3 showed that after treatment with glucoamylase the amount of hydrolysis
product disappeared, only the spot of glucose and isobutyl monoglucoside increased due to the glycosidic
linkage was hydrolyzed except those between alcohol and glucose. The reaction mixture was further treated
with α-glucosidase to elucidate the linkage between alcohol and glucose. It can be been that the spot of
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isobutyl glucoside disappeared (Fig. 3, Lane 8) which could be due to the α-linkage was hydrolyzed. Thus, it
seems likely that CGTase could catalyze transglycosylation reaction from β-CD to alcohols.
Fig. 2: TLC analysis of the transglycosylation products
from β-CD to various alcohols by CGTase. Lane 1-4
standard β-CD, glucose, maltose and maltotrios, Lane 5
reaction mixture without alcohol acceptor, Lane 6-7
reaction mixture with 30% (v/v) methanol and ethanol,
Lane 8-13 reaction mixture with 10% (v/v) 1-propanol,
2-propanol, 1-butanol, isobutanol, 2-butanol and tertbutanol, Lane 14-15 reaction mixture with 5% (v/v)
pentanol and 2-pentanol, Lane 16-17 reaction mixture
with 2% (v/v) 2-hexanol and cyclohexanol, Lane 18-19
reaction mixture with 1% (v/v) 1-heptanol and 1octanol.
Fig. 3: TLC chromatogram of amylolytic enzyme
treatment of CGTase reaction products. Lane 1-4
standard glucose, maltose, maltotriose and methyl
monoglucoside Lane 5 reaction mixture at 0 minute
incubation, Lane 6 reaction mixture after 24 hours
incubation, Lane 7 reaction mixture (24 hours) treated
with glucoamylase, Lane 8 reaction mixture (24 hours)
with -glucosidase and Lane 9 reaction mixture (24
hours) with glucoamylase and -glucosidase, Lane 1013 standard methyl monoglucoside, glucose, maltose
and maltotriose.
3.3. Donor Specificity
CGTase could use various types of carbohydrate sources as a glycosyl donor. The products obtained
from all donors were comparable as shown in Table1. Raw starch was successfully used as a glycosyl donor
with similarity in product types and the amount obtained. Thus it might have high potential for the
application in the manufacturing process because it is inexpensive and could be easily obtained from
renewable agricultural resource.
Table 1: The amount of alkyl glycoside products from various glycosyl donors
Donors
Total production yield from various donors (mol %)*
Isobutyl
Isobutyl
Isobutyl
monoglucoside
maltoside
maltotrioside
26.4
21.9
11.8
60.2
24.6
21.9
10.0
56.5
26.4
21.9
10.0
58.3
22.8
18.2
10.0
51.0
β-CD
Soluble stach
Flomax
®
Raw starch
Total
*mole % represent the mole percent of alkyl glycosides spot relative to the total mole present in the β-CD substrate (0.6% w/v) each values were from
separate experiment and quantitated using methyl-α-D-glucopyranolside as standard.
4. Acknowledgements
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This research was supported by Ratchadapiseksompote Research Fund granted to Starch and
Cyclodextrin Research Unit, Department of Biochemistry, Faculty of Science, Chulalongkorn University.
The author would also like to thank Chulalongkorn University Graduate Scholarship to commemorate the
72nd Anniversary of his Majesty King Bhumibol Adulyadej.
5. References
[1] C. Baron, and T.E. Thompson. Solubilization of bacterial membrane proteins using alkyl glucosides and
dioctanoyl phosphotidylcholine. Biochim et Biophy Acta. 1975, 382: 276-285.
[2] A. Ismail, S. Soultani , and M. Ghoul. Enzymatic-catalyzed synthesis of alkylglycosides in monophasic and
biphasic systems. I. The transglycosylation reaction. J Biotechno. 1999, 69: 135- 143.
[3]
M.P. Bousquet, R.M. Willemot, P.F. Monsan, F. Paul and E. Boures. Enzymatic synthesis of α-butylglycoside in
a biphasic butanol-water system using the α-tranglucosidase from Aspergillus niger. Method in Biotechnol. 10:
291-296.
[4] J. Kosary, E.S. Banyai and L. Boross. Reverse hydrolytic process for O-alkylation of glucose catalysed by
immobilized α- and β-glucosidases. J.Biotechnology. 1998, 66:83-86.
[5] D.B. Sarney, and E.N. Vulfson. Application of enzymes to the synthesis of surfactants. Trends in Biotechnol. 1995,
13: 164-172.
[6] K. Chotipanang, W. Bhunthumnavin , and M.H. Prousoontorn. Synthesis of alkyl glycosides from cyclodextrin
using cyclodextrin glycosyltransferase from Paenibacillus sp. RB01. J Incl Phenom and Macrocycl Chem. 2011,
70: 359-368.
[7] K. Kato, and K. Horikoshi. Immobilized cyclodextrin glucanotransferase of an alkolophilic Bacillus sp. No 38-2.
Biotechnol. Bioeng. 1984, 26: 595-598.
[8] A. Goel, N. Nene. Modifications in the phenolphthalein method for spectrophotometric estimation of beta
cyclodextrin. Starch. 1995, 47: 399-400.
[9] Y. Liu, Y. Chen, S.Z. Kang, L. Li, C. H. Diao and H.Y. Zhang. Synthesis of novel benzo-15-crown-5-tethered βcyclodextrins and their enhanced molecular binding abilities by alkali metal cation coordination. J Incl Phenom
and Macrocycl Chem. 2003, 47: 91-95.
[10] K. Lirtprapamongkol and J. Svasti. Alkyl glucoside synthesis using Thai rosewood β-glucosidase. Biotechnology
let. 2000, 22: 1889-1894.
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